Wheat plants having increased resistance to imidazolinone herbicides

ABSTRACT

The present invention is directed to wheat plants having increased resistance to an imidazolinone herbicide. More particularly, the present invention includes wheat plants containing one or more IMI nucleic acids such as a Gunner IMI 205, Gunner IMI 208 and Madsen IMI cultivar. The present invention also includes seeds produced by these wheat plants and methods of controlling weeds in the vicinity of these wheat plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/362,935, filed Jan. 30, 2009, now U.S. Pat. No. 8,389,809, to beissued on Mar. 5, 2013, which is a divisional of U.S. patent applicationSer. No. 10/486,595, filed Sep. 13, 2004, now U.S. Pat. No. 7,528,397,issued on May 5, 2009, which is the U.S. National Stage of InternationalApplication PCT/US02/21753, filed Jul. 10, 2002, which published inEnglish on Feb. 20, 2003 and designates the U.S., which claims thepriority benefit of U.S. Provisional Application Ser. No. 60/311,141filed Aug. 9, 2001; all of which are hereby incorporated herein in theirentirety by reference.

FIELD OF THE INVENTION

The present invention relates in general to plants having an increasedresistance to imidazolinone herbicides. More specifically, the presentinvention relates to wheat plants obtained by mutagenesis andcross-breeding and transformation that have an increased resistance toimidazolinone herbicides.

BACKGROUND OF THE INVENTION

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) is the first enzyme thatcatalyzes the biochemical synthesis of the branched chain amino acidsvaline, leucine and isoleucine (Singh B. K., 1999 Biosynthesis ofvaline, leucine and isoleucine in: Singh B. K. (Ed) Plant amino acids.Marcel Dekker Inc. New York, N.Y. Pg 227-247). AHAS is the site ofaction of four structurally diverse herbicide families including thesulfonylureas (LaRossa R A and Falco S C, 1984 Trends Biotechnol2:158-161), the imidazolinones (Shaner et al., 1984 Plant Physiol76:545-546), the triazolopyrimidines (Subramanian and Gerwick, 1989Inhibition of acetolactate synthase by triazolopyrimidines in (ed)Whitaker J R, Sonnet P E Biocatalysis in agricultural biotechnology. ACSSymposium Series, American Chemical Society. Washington, D.C. Pg277-288), and the pyrimidyloxybenzoates (Subramanian et al., 1990 PlantPhysiol 94: 239-244.). Imidazolinone and sulfonylurea herbicides arewidely used in modern agriculture due to their effectiveness at very lowapplication rates and relative non-toxicity in animals. By inhibitingAHAS activity, these families of herbicides prevent further growth anddevelopment of susceptible plants including many weed species. Severalexamples of commercially available imidazolinone herbicides are PURSUIT®(imazethapyr), SCEPTER® (imazaquin) and ARSENAL® (imazapyr). Examples ofsulfonylurea herbicides are chlorsulfuron, metsulfuron methyl,sulfometuron methyl, chlorimuron ethyl, thifensulfuron methyl,tribenuron methyl, bensulfuron methyl, nicosulfuron, ethametsulfuronmethyl, rimsulfuron, triflusulfuron methyl, triasulfuron, primisulfuronmethyl, cinosulfuron, amidosulfuron, fluzasulfuron, imazosulfuron,pyrazosulfuron ethyl and halosulfuron.

Due to their high effectiveness and low-toxicity, imidazolinoneherbicides are favored for application by spraying over the top of awide area of vegetation. The ability to spray an herbicide over the topof a wide range of vegetation decreases the costs associated withplantation establishment and maintenance and decreases the need for sitepreparation prior to use of such chemicals. Spraying over the top of adesired tolerant species also results in the ability to achieve maximumyield potential of the desired species due to the absence of competitivespecies. However, the ability to use such spray-over techniques isdependent upon the presence of imidazolinone resistant species of thedesired vegetation in the spray over area.

Among the major agricultural crops, some leguminous species such assoybean are naturally resistant to imidazolinone herbicides due to theirability to rapidly metabolize the herbicide compounds (Shaner andRobinson, 1985 Weed Sci. 33:469-471). Other crops such as corn (Newhouseet al., 1992 Plant Physiol. 100:882-886) and rice (Barrette et al., 1989Crop Safeners for Herbicides, Academic Press New York, pp. 195-220) aresomewhat susceptible to imidazolinone herbicides. The differentialsensitivity to the imidazolinone herbicides is dependent on the chemicalnature of the particular herbicide and differential metabolism of thecompound from a toxic to a non-toxic form in each plant (Shaner et al.,1984 Plant Physiol. 76:545-546; Brown et al., 1987 Pestic. Biochm.Physiol. 27:24-29). Other plant physiological differences such asabsorption and translocation also play an important role in sensitivity(Shaner and Robinson, 1985 Weed Sci. 33:469-471).

Crop cultivars resistant to imidazolinones, sulfonylureas andtriazolopyrimidines have been successfully produced using seed,microspore, pollen, and callus mutagenesis in Zea Mays, Arabidopsisthaliana, Brassica napus, Glycine max, and Nicotiana tabacum (Sebastianet al., 1989 Crop Sci. 29:1403-1408; Swanson et al., 1989 Theor. Appl.Genet. 78:525-530; Newhouse et al., 1991 Theor. Appl. Genet 83:65-70;Sathasivan et al., 1991 Plant Physiol. 9 7:1 0 4 4-1050; Mourand et al.,1993 J. Heredity 84:91-96). In all cases, a single, partially dominantnuclear gene conferred resistance. Four imidazolinone resistant wheatplants were also previously isolated following seed mutagenesis ofTriticum aestivum L. cv Fidel (Newhouse et al., 1992 Plant Physiol.100:882-886). Inheritance studies confirmed that a single, partiallydominant gene conferred resistance. Based on allelic studies, theauthors concluded that the mutations in the four identified lines werelocated at the same locus. One of the Fidel cultivar resistance geneswas designated FS-4 (Newhouse et al., 1992 Plant Physiol. 100:882-886).

Computer-based modeling of the three dimensional conformation of theAHAS-inhibitor complex predicts several amino acids in the proposedinhibitor binding pocket as sites where induced mutations would likelyconfer selective resistance to imidazolinones (Ott et al., 1996 J. Mol.Biol. 263:359-368) Wheat plants produced with some of these rationallydesigned mutations in the proposed binding sites of the AHAS enzyme havein fact exhibited specific resistance to a single class of herbicides(Ott et al., 1996 J. Mol. Biol. 263:359-368).

Plant resistance to imidazolinone herbicides has also been reported in anumber of patents. U.S. Pat. Nos. 4,761,373, 5,331,107, 5,304,732,6,211,438, 6,211,439 and 6,222,100 generally describe the use of analtered AHAS gene to elicit herbicide resistance in plants, andspecifically discloses certain imidazolinone resistant corn lines. U.S.Pat. No. 5,013,659 discloses plants exhibiting herbicide resistancepossessing mutations in at least one amino acid in one or more conservedregions. The mutations described therein encode either cross-resistancefor imidazolinones and sulfonylureas or sulfonylurea-specificresistance, but imidazolinone-specific resistance is not described.Additionally, U.S. Pat. Nos. 5,731,180 and 5,767,361 discuss an isolatedgene having a single amino acid substitution in a wild-type monocot AHASamino acid sequence that results in imidazolinone-specific resistance.

To date, the prior art has not described imidazolinone resistant wheatplants containing more than one altered AHAS gene. Nor has the prior artdescribed imidazolinone resistant wheat plants containing mutations ongenomes other than the genome from which the FS-4 gene is derived.Therefore, what is needed in the art is the identification ofimidazolinone resistance genes from additional genomes. What are alsoneeded in the art are wheat plants having increased resistance toherbicides such as imidazolinone and containing more than one alteredAHAS gene. Also needed are methods for controlling weed growth in thevicinity of such wheat plants. These compositions and methods wouldallow for the use of spray over techniques when applying herbicides toareas containing wheat plants.

SUMMARY OF THE INVENTION

The present invention provides wheat plants comprising IMI nucleicacids, wherein the wheat plant has increased resistance to animidazolinone herbicide as compared to a wild-type variety of the plant.The wheat plants can contain one, two, three or more IMI nucleic acids.In one embodiment, the wheat plant comprises multiple IMI nucleic acidslocated on different genomes. Preferably, the IMI nucleic acids encodeproteins comprising a mutation in a conserved amino acid sequenceselected from the group consisting of a Domain A, a Domain B, a DomainC, a Domain D and a Domain E. More preferably, the mutation is in aconserved Domain E or a conserved Domain C. Also provided are plantparts and plant seeds derived from the wheat plants described herein. Inanother embodiment, the wheat plant comprises an IMI nucleic acid thatis not an Imi1 nucleic acid. The IMI nucleic acid can be an Imi2 or Imi3nucleic acid, for example.

The IMI nucleic acids of the present invention can comprise a nucleotidesequence selected from the group consisting of: a polynucleotide of SEQID NO:1; a polynucleotide of SEQ ID NO:3; a polynucleotide of SEQ IDNO:5; a polynucleotide comprising at least 60 consecutive nucleotides ofany of the aforementioned polynucleotides; and a polynucleotidecomplementary to any of the aforementioned polynucleotides.

The plants of the present invention can be transgenic or non-transgenic.Examples of non-transgenic wheat plants having increased resistance toimidazolinone herbicides include a wheat plant having an ATCC PatentDeposit Designation Number PTA-4213, PTA-4214 or PTA-4255; or a mutant,recombinant, or genetically engineered derivative of the plant with ATCCPatent Deposit Designation Number PTA-4213, PTA-4214 or PTA-4255; or ofany progeny of the plant with ATCC Patent Deposit Designation NumberPTA-4213, PTA-4214 or PTA-4255; or a plant that is a progeny of any ofthese plants.

In addition to the compositions of the present invention, severalmethods are provided. Described herein are methods of modifying aplant's tolerance to an imidazolinone herbicide comprising modifying theexpression of an IMI nucleic acid in the plant. Also described aremethods of producing a transgenic plant having increased tolerance to animidazolinone herbicide comprising, transforming a plant cell with anexpression vector comprising one or more IMI nucleic acids andgenerating the plant from the plant cell. The invention further includesa method of controlling weeds within the vicinity of a wheat plant,comprising applying an imidazolinone herbicide to the weeds and to thewheat plant, wherein the wheat plant has increased resistance to theimidazolinone herbicide as compared to a wild type variety of the wheatplant and wherein the plant comprises one or more IMI nucleic acids. Insome preferred embodiments of these methods, the plants comprisemultiple IMI nucleic acids that are located on different wheat genomes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the conserved amino acidsequences in the AHAS genes implicated in resistance to various AHASinhibitors. The specific amino acid site responsible for resistance isindicated by an underline. (Modified from Devine, M. D. and Eberlein, C.V., 1997 Physiological, biochemical and molecular aspects of herbicideresistance based on altered target sites in Herbicide Activity:Toxicity, Biochemistry, and Molecular Biology, IOS Press Amersterdam, p.159-185).

FIGS. 2A-C are tables showing the inhibition of AHAS enzyme activity inwild-type wheat (variety Gunner), AP205CL (FIG. 2A), AP602CL (FIG. 2B)and Madsen1 (FIG. 2C) by imidazolinone herbicide imazamox. Values areexpressed as a percent of uninhibited activity.

FIG. 3 is a table showing the decreased injury of Madsen1 by imazamox ascompared to a Tea1 wheat control.

FIGS. 4A-B are tables showing the feedback inhibition of AHAS enzymeactivity by leucine and valine in wild-type wheat (variety Gunner),AP205CL (FIG. 4A) and AP602CL (FIG. 4B).

FIG. 5A depicts the partial nucleic acid sequences of the Gunner 208-30wheat variety (SEQ ID:12), and the wild-type wheat variety, wt19(SEQID:13). FIG. 5B depicts the corresponding partial amino acid sequencesof the Gunner 208-30 wheat variety (SEQ ID:14) and the wild-type wheatvariety, wt19(SEQ ID:15).

DETAILED DESCRIPTION

The present invention is directed to wheat plants, wheat plant parts andwheat plant cells having increased resistance to imidazolinoneherbicides. The present invention also includes seeds produced by thewheat plants described herein and methods for controlling weeds in thevicinity of the wheat plants described herein. It is to be understoodthat as used in the specification and in the claims, “a” or “an” canmean one or more, depending upon the context in which it is used. Thus,for example, reference to “a cell” can mean that at least one cell canbe utilized.

As used herein, the term “wheat plant” refers to a plant that is amember of the Triticum genus. The wheat plants of the present inventioncan be members of a Triticum genus including, but not limited to, T.aestivum, T. turgidum, T timopheevii, T. monococcum, T. zhukovskyi andT. urartu and hybrids thereof. Examples of T aestivum subspeciesincluded within the present invention are aestivum (common wheat),compactum (club wheat), macha (macha wheat), vavilovi (vavilovi wheat),spelta and sphaecrococcum (shot wheat). Examples of T. turgidumsubspecies included within the present invention are turgidum,carthlicum, dicoccon, durum, paleocolchicum, polonicum, turanicum anddicoccoides. Examples of T. monococcum subspecies included within thepresent invention are monococcum (einkorn) and aegilopoides. In oneembodiment of the present invention, the wheat plant is a member of theTriticum aestivum L. species, and more particularly, a Gunner or Madsencultivar.

The term “wheat plant” is intended to encompass wheat plants at anystage of maturity or development as well as any tissues or organs (plantparts) taken or derived from any such plant unless otherwise clearlyindicated by context. Plant parts include, but are not limited to,stems, roots, flowers, ovules, stamens, leaves, embryos, meristematicregions, callus tissue, anther cultures, gametophytes, sporophytes,pollen, microspores, protoplasts and the like. The present inventionalso includes seeds produced by the wheat plants of the presentinvention. In one embodiment, the seeds are true, breeding for anincreased resistance to an imidazolinone herbicide as compared to a wildtype variety of the wheat plant seed.

The present invention describes a wheat plant comprising one or more IMInucleic acids, wherein the wheat plant has increased resistance to animidazolinone herbicide as compared to a wild-type variety of the plant.As used herein, the term “IMI nucleic acid” refers to a nucleic acidthat is mutated from an AHAS nucleic acid in a wild type wheat plantthat confers increased imidazolinone resistance to a plant in which itis transcribed. In one embodiment, the wheat plant comprises multipleIMI nucleic acids. As used when describing the IMI nucleic acids, theterm “multiple” refers to IMI nucleic acids that have differentnucleotide sequences and does not refer to a mere increase in number ofthe same IMI nucleic acid. For example, the IMI nucleic acids can bedifferent due to the fact that they are derived from or located ondifferent wheat genomes.

It is possible for the wheat plants of the present invention to havemultiple IMI nucleic acids from different genomes since these plants cancontain more than one genome. For example, a Triticum aestivum wheatplant contains three genomes sometimes referred to as the A, B and Dgenomes. Because AHAS is a required metabolic enzyme, it is assumed thateach genome has at least one gene coding for the AHAS enzyme, commonlyseen with other metabolic enzymes in hexaploid wheat that have beenmapped. The AHAS nucleic acid on each genome can, and usually does,differ in its nucleotide sequence from an AHAS nucleic acid on anothergenome. One of skill in the art can determine the genome of origin ofeach AHAS nucleic acid through genetic crossing and/or either,sequencing methods or exonuclease digestion methods known to those ofskill in the art and as also described in Example 2 below. For thepurposes of this invention, IMI nucleic acids derived from one of the A,B or D genomes are distinguished and designated as Imi1, Imi2 or Imi3nucleic acids. It is not stated herein that any particular Imi nucleicacid class correlates with any particular A, B or D genome. For example,it is not stated herein that the Imi1 nucleic acids correlate to Agenome nucleic acids, that Imi2 nucleic acids correlate to B genomenucleic acids, etc. The Imi1, Imi2 and Imi3 designations merely indicatethat the IMI nucleic acids within each such class do not segregateindependently, whereas two IMI nucleic acids from different classes dosegregate independently and may therefore be derived from differentwheat genomes.

The Imi1 class of nucleic acids includes the FS-4 gene as described byNewhouse et al. (1992 Plant Physiol. 100:882-886) and the Gunner IMI1205 gene described in more detail below. The Imi2 class of nucleic acidsincludes the Gunner IMI2 208 gene and the Madsen IMI2 gene describedbelow. As shown from the members of the Imi1 class of nucleic acids,each Imi class can include members from different wheat species.Therefore, each Imi class includes IMI nucleic acids that differ intheir nucleotide sequence but that are nevertheless designated asoriginating from, or being located on, the same wheat genome usinginheritance studies as known to those of ordinary skill in the art.

Accordingly, the present invention includes a wheat plant comprising oneor more IMI nucleic acids, wherein the wheat plant has increasedresistance to an imidazolinone herbicide as compared to a wild-typevariety of the plant and wherein the one or more IMI nucleic acids areselected from a group consisting of an Imi1, Imi2 and Imi3 nucleic acid.In one embodiment, the plant comprises an Imi1 nucleic and an Imi2nucleic acid. In a preferred embodiment, the Imi1 nucleic acid comprisesthe polynucleotide sequence shown in SEQ ID NO:1 and the Imi2 nucleicacid comprises the polynucleotide sequence shown in SEQ ID NO:3 or SEQID NO:5. In another embodiment, the plant comprises an Imi3 nucleicacid.

As used herein with regard to nucleic acids, the term “from” refers to anucleic acid “located on” or “derived from” a particular genome. Theterm “located on refers to a nucleic acid contained within thatparticular genome. As also used herein with regard to a genome, the term“derived from” refers to a nucleic acid that has been removed orisolated from that genome. The term “isolated” is defined in more detailbelow.

In another embodiment, the wheat plant comprises an IMI nucleic acid,wherein the nucleic acid is a non-Imi1 nucleic acid. The term“non-Imi1”, refers to an IMI nucleic acid that is not a member of theImi1 class as described above. Examples of non-Imi1 nucleic acid are thepolynucleotide sequences shown in SEQ ID NO:3 and SEQ. NO:5.Accordingly, in a preferred embodiment, the wheat plant comprises an IMInucleic acid comprising the polynucleotide sequence shown in SEQ ID NO:3or SEQ ID NO:5.

The present invention includes wheat plants comprising one, two, threeor more IMI nucleic acids, wherein the wheat plant has increasedresistance to an imidazolinone herbicide as compared to a wild-typevariety of the plant. The IMI nucleic acids can comprise a nucleotidesequence selected from the group consisting of a polynucleotide of SEQID NO:1; a polynucleotide of SEQ ID NO:3; a polynucleotide of SEQ IDNO:5; a polynucleotide encoding a polypeptide of SEQ ID NO:2, apolynucleotide encoding a polypeptide of SEQ ID NO:4; a polynucleotideencoding a polypeptide of SEQ ID NO:6; a polynucleotide comprising atleast 60 consecutive nucleotides of any of the aforementionedpolynucleotides; and a polynucleotide complementary to any of theaforementioned polynucleotides.

The imidazolinone herbicide can be selected from, but is not limited to,PURSUIT® (imazethapyr), CADRE® (imazapic), RAPTOR® (imazamox), SCEPTER®(imazaquin), ASSERT® (imazethabenz), ARSENAL® (imazapyr), a derivativeof any of the aforementioned herbicides, or a mixture of two or more ofthe aforementioned herbicides, for example, imazapyr/imazamox(ODYSSEY®). More specifically, the imidazolinone herbicide can beselected from, but is not limited to,2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acid,2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-3-quinolinecarboxylicacid, 5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinicacid,2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinicacid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinicacid, and a mixture of methyl6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-m-toluate and methyl2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-toluate. The use of5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-nicotinic acidand2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinicacid is preferred. The use of2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-(methoxymethyl)-nicotinicacid is particularly preferred.

In one embodiment, the wheat plant comprises two IMI nucleic acids,wherein the nucleic acids are derived from or located on different wheatgenomes. Preferably, the two nucleic acids are an Imi1 nucleic acid andan Imi2 nucleic acid. More preferably, the Imi1 nucleic acid comprisesthe polynucleotide sequence of SEQ ID NO:1 and the Imi2 nucleic acidcomprises the polynucleotide sequence of SEQ ID NO:3 or SEQ ID NO:5. Inanother embodiment, the wheat plant comprises one IMI nucleic acid,wherein the nucleic acid comprises the polynucleotide sequence of SEQ IDNO:1, SEQ ID NO:3 or SEQ ID NO:5. In yet another embodiment, the wheatplant comprises three or more IMI nucleic acids wherein each nucleicacid is from a different genome. Preferably, at least one of the threeIMI nucleic acids comprises a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5.

In a preferred embodiment of the present invention, the one or more IMInucleic acids contained within the plant encode an amino acid sequencecomprising a mutation in a domain that is conserved among several AHASproteins. These conserved domains are referred to herein as Domain A,Domain B , Domain C, Domain D and Domain E. FIG. 1 shows the generallocation of each domain in an AHAS protein. Domain A contains the aminoacid sequence AITGQVPRRMIGT (SEQ ID NO:7). Domain B contains the aminoacid sequence QWED (SEQ ID NO:8). Domain C contains the amino acidsequence VFAYPGGASMEIHQALTRS (SEQ ID NO:9). Domain D contains the aminoacid sequence AFQETP (SEQ ID NO:10). Domain E contains the amino acidsequence IPSGG (SEQ ID NO:11). The present invention also contemplatesthat there may be slight variations in the conserved domains, forexample; in cockleberry plants, the serine residue in Domain E isreplaced by an alanine residue.

Accordingly, the present invention includes a wheat plant comprising anIMI nucleic acid that encodes an amino acid sequence having a mutationin a conserved domain selected from the group consisting of a Domain A,a Domain B, a Domain C, a Domain D and a Domain E. In one embodiment,the wheat plant comprises an IMI nucleic acid that encodes an amino acidsequence having a mutation in a Domain E. In further preferredembodiments, the mutations in the conserved domains occur at thelocations indicated by the following underlining: AITGQVPRRMIGT (SEQ IDNO:7); QWED (SEQ ID NO:8); VFAYPGGASMEIHQALTRS (SEQ ID NO:9); AFQETP(SEQ ID NO:10) and IPSGG (SEQ ID NO:11). One preferred substitution isasparagine for serine in Domain E (SEQ ID NO:11).

The wheat plants described herein can be either transgenic wheat plantsor non-transgenic wheat plants. As used herein, the term “transgenic”refers to any plant, plant cell, callus, plant tissue or plant part,that contains all or part of at least one recombinant polynucleotide. Inmany cases, all or part of the recombinant polynucleotide is stablyintegrated into a chromosome or stable extra-chromosomal element, sothat it is passed on to successive generations. For the purposes of theinvention, the term “recombinant polynucleotide” refers to apolynucleotide that has been altered, rearranged or modified by geneticengineering. Examples include any cloned polynucleotide, orpolynucleotides, that are linked or joined to heterologous sequences.The term “recombinant” does not refer to alterations of polynucleotidesthat result from naturally occurring events, such as spontaneousmutations, or from non-spontaneous mutagenesis followed by selectivebreeding. Plants containing mutations arising due to non-spontaneousmutagenesis and selective breeding are referred to herein asnon-transgenic plants and are included in the present invention. Inembodiments wherein the wheat plant is transgenic and comprises multipleIMI nucleic acids, the nucleic acids can be derived from differentgenomes or from the same genome. Alternatively, in embodiments whereinthe wheat plant is non-transgenic and comprises multiple IMI nucleicacids, the nucleic acids are located on different genomes or on the samegenome.

An example of a non-transgenic wheat plant cultivar comprising one IMInucleic acid is the plant cultivar deposited with the ATCC under PatentDeposit Designation Number PTA-4213 and designated herein as the GunnerIMI 205 wheat cultivar. The Gunner IMI 205 wheat cultivar contains anImi1 nucleic acid. The partial nucleotide sequences corresponding to theGunner IMI1 205 gene is shown in SEQ ID NO:1.

Another example of a non-transgenic wheat plant cultivar comprising oneIMI nucleic acid is the plant cultivar deposited with the ATCC underPatent Deposit Designation Number PTA-4214 and designated herein as theGunner IMI 208 wheat cultivar. The Gunner IMI 208 wheat cultivarcontains an Imi2 nucleic acid. The partial nucleotide sequencecorresponding to the Gunner IMI2 208 gene is shown in SEQ ID NO:3.

Yet another example of a non-transgenic wheat plant cultivar comprisingone IMI nucleic acid is the plant cultivar deposited with the ATCC underPatent Deposit Designation Number PTA-4255 and designated herein as theMadsen IMI wheat cultivar. The Madsen IMI wheat cultivar contains anImi2 nucleic acid. The partial nucleotide sequence corresponding to theMadsen 1MI2 gene is shown in SEQ ID NO:5.

Separate deposits of 2500 seeds of the Gunner IMI 205, Gunner IMI 208and Madsen IMI wheat cultivars were made with the American Type CultureCollection, Manassas, Va. on Apr. 9, 2002 (Gunner IMI 205 and Gunner IMI208) and on May 1, 2002 (Madsen IMI). These deposits were made inaccordance with the terms and provisions of the Budapest Treaty relatingto the deposit of microorganisms. The deposits were made for a term ofat least thirty years and at least five years after the most recentrequest for the furnishing of a sample of the deposit is received by theATCC. The deposited seeds were accorded Patent Deposit DesignationNumbers PTA-4213 (Gunner IMI 205), PTA-4214 (Gunner IMI 208) andPTA-4255 (Madsen IMI).

The present invention includes the wheat plant having a Patent DepositDesignation Number PTA-4213, PTA-4214 or PTA-4255; a mutant,recombinant, or genetically engineered derivative of the plant withPatent Deposit Designation Number PTA-4213, PTA-4214 or PTA-4255; anyprogeny of the plant with Patent Deposit Designation Number PTA-4213,PTA-4214 or PTA-4255; and a plant that is the progeny of any of theseplants. In a preferred embodiment, the wheat plant of the presentinvention additionally has the herbicide resistance characteristics ofthe plant with Patent Deposit Designation Number PTA-4213, PTA-4214 orPTA-4255.

Also included in the present invention are hybrids of the Gunner IMI205, Gunner IMI 208 or Madsen IMI wheat cultivars described herein andanother wheat cultivar. The other wheat cultivar includes, but is notlimited to, T. aestivum L. cv Fidel and any wheat cultivar harboring amutant gene FS-1, FS-2, FS-3 or FS-4. (See U.S. Pat. No 6,339,184 andU.S. patent application Ser. No. 08/474,832). Preferred hybrids containa combination of Imi1, Imi2 and/or Imi3 nucleic acids. Examples ofpreferred hybrids are Gunner IMI 205/Gunner IMI 208 hybrids. The GunnerIMI 205/Gunner IMI 208 hybrids comprise an Imi1 nucleic acid and an Imi2nucleic acid.

The terms “cultivar” and “variety” refer to a group of plants within aspecies defined by the sharing of a common set of characteristics ortraits accepted by those skilled in the art as sufficient to distinguishone cultivar or variety from another cultivar or variety. There is noimplication in either term that all plants of any given cultivar orvariety will be genetically identical at either the whole gene ormolecular level or that any given plant will be homozygous at all loci.A cultivar or variety is considered “true breeding” for a particulartrait if, when the true-breeding cultivar or variety is self-pollinated,all of the progeny contain the trait. In the present invention, thetrait arises from a mutation in an AHAS gene of the wheat plant or seed.

It is to be understood that the wheat plant of the present invention cancomprise a wild type or non-mutated AHAS gene in addition to an IMIgene. As described in Examples 1 and 2, it is contemplated that theGunner IMI 205, Gunner IMI 208 and Madsen IMI wheat cultivars contain amutation in only one of multiple AHAS isoenzymes. Therefore, the presentinvention includes a wheat plant comprising one or more IMI nucleicacids in addition to one or more wild type or non-mutated AHAS nucleicacids.

In addition to wheat plants, the present invention encompasses isolatedIMI proteins and nucleic acids. The nucleic acids comprise apolynucleotide selected from the group consisting of a polynucleotide ofSEQ ID NO:1; a polynucleotide of SEQ ID NO:3; a polynucleotide of SEQ IDNO:5; a polynucleotide encoding a polypeptide of SEQ ID NO:2; apolynucleotide encoding a polypeptide of SEQ ID NO:4; a polynucleotideencoding a polypeptide of SEQ ID NO:6; a polynucleotide comprising atleast 60 consecutive nucleotides of any of the aforementionedpolynucleotides; and a polynucleotide complementary to any of theaforementioned polynucleotides. In a preferred embodiment, the IMInucleic acid comprises a polynucleotide sequence of SEQ ID NO:1, SEQ IDNO:3 or SEQ ID NO:5.

The term “AHAS protein” refers to an acetohydroxyacid synthase proteinand the term “IMI protein” refers to any AHAS protein that is mutatedfrom a wild type AHAS protein and that confers increased imidazolinoneresistance to a plant, plant cell, plant part, plant seed or planttissue when it is expressed therein. In a preferred embodiment, the IMIprotein comprises a polypeptide encoded by a polynucleotide sequencecomprising SEQ ID NO:1, SEQ ID NO:3 or SEQ ID. NO:5. As also usedherein, the terms “nucleic acid” and “polynucleotide” refer to RNA orDNA that is linear or branched, single or double stranded, or a hybridthereof. The term also encompasses RNA/DNA hybrids. These terms alsoencompass untranslated sequence located at both the 3′ and 5′ ends ofthe coding region of the gene: at least about 1000 nucleotides ofsequence upstream from the 5′ end of the coding region and at leastabout 200 nucleotides of sequence downstream from the 3′ end of thecoding region of the gene. Less common bases, such as inosine,5-methylcytosine, 6-methyladenine, hypoxanthine and others can also beused for antisense, dsRNA and ribozyme pairing. For example,polynucleotides that contain C-5 propyne analogues of uridine andcytidine have been shown to bind RNA with high affinity and to be potentantisense inhibitors of gene expression. Other modifications, such asmodification to the phosphodiester backbone, or the 2′-hydroxy in theribose sugar group of the RNA can also be made. The antisensepolynucleotides and ribozymes can consist entirely of ribonucleotides,or can contain mixed ribonucleotides and deoxyribonucleotides. Thepolynucleotides of the invention may be produced by any means, includinggenomic preparations, cDNA preparations, in vitro synthesis, RT-PCR andin vitro or in vivo transcription.

An “isolated” nucleic acid molecule is one that is substantiallyseparated from other nucleic acid molecules, which are present in thenatural source of the nucleic acid (i.e., sequences encoding otherpolypeptides).

Preferably, an “isolated” nucleic acid is free of some of the sequencesthat naturally flank the nucleic acid (i.e., sequences located at the 5′and 3′ ends of the nucleic acid) in its naturally occurring replicon.For example, a cloned nucleic acid is considered isolated. In variousembodiments, the isolated IMI nucleic acid molecule can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotidesequences which naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived (e.g., a Triticumaestivum cell). A nucleic acid is also considered isolated if it hasbeen altered by human intervention, or placed in a locus or locationthat is not its natural site, or if it is introduced into a cell byagroinfection or biolistics. Moreover, an “isolated” nucleic acidmolecule, such as a cDNA molecule, can be free from some of the othercellular material with which it is naturally associated, or culturemedium when produced by recombinant techniques, or chemical precursorsor other chemicals when chemically synthesized.

Specifically excluded from the definition of “isolated nucleic acids”are: naturally-occurring chromosomes (such as chromosome spreads),artificial chromosome libraries, genomic libraries, and cDNA librariesthat exist either as an in vitro nucleic acid preparation or as atransfected/transformed host cell preparation, wherein the host cellsare either an in vitro heterogeneous preparation or plated as aheterogeneous population of single colonies. Also, specifically excludedare the above libraries wherein a specified nucleic acid makes up lessthan 5% of the number of nucleic acid inserts in the vector molecules.Further specifically excluded are whole cell genomic DNA or whole cellRNA preparations (including whole cell preparations that aremechanically sheared or enzymatically digested). Even furtherspecifically excluded are the whole cell preparations found as either anin vitro preparation or as a heterogeneous mixture separated byelectrophoresis wherein the nucleic acid of the invention has notfurther been separated from the heterologous nucleic acids in theelectrophoresis medium (e.g., further separating by excising a singleband from a heterogeneous band population in an agarose gel or nylonblot).

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule containing a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:5 or a portion thereof, can be isolated using standardmolecular biology techniques and the sequence information providedherein. For example, a T. aestivum IMI cDNA can be isolated from a Taestivum library using all or a portion of the sequence of SEQ ID NO:1,SEQ ID NO:3 or SEQ ID. NO:5. Moreover, a nucleic acid moleculeencompassing all or a portion of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5can be isolated by the polymerase chain reaction using oligonucleotideprimers designed based upon this sequence. For example, mRNA can beisolated from plant cells (e.g., by the guanidinium-thiocyanateextraction procedure of Chirgwin et al., 1979 Biochemistry 18:5294-5299)and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLVreverse transcriptase, available from Gibco/BRL, Bethesda, Md.; or AMVreverse′ transcriptase, available from Seikagaku America, Inc., St.Petersburg, Fla.). Synthetic oligonucleotide primers for polymerasechain reaction amplification can be designed based upon the nucleotidesequence shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5. A nucleicacid molecule of the invention can be amplified using cDNA or,alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid molecule so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to an IMI nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

The IMI nucleic acids of the present invention can comprise sequencesencoding an IMI protein (i.e., “coding regions”), as well as 5′untranslated sequences and 3′ untranslated sequences. Alternatively, thenucleic acid molecules of the present invention can comprise only thecoding regions of an IMI gene, or can contain whole genomic fragmentsisolated from genomic DNA. A coding region of these sequences isindicated as an “ORF position”. Moreover, the nucleic acid molecule ofthe invention can comprise a portion of a coding region of an IMI gene,for example, a fragment that can be used as a probe or primer. Thenucleotide sequences determined from the cloning of the IMI genes from Taestivum allow for the generation of probes and primers designed for usein identifying and/or cloning IMI homologs in other cell types andorganisms, as well as IMI homologs from other wheat plants and relatedspecies. The portion of the coding region can also encode a biologicallyactive fragment of an IMI protein.

As used herein, the term “biologically active portion of an IMI proteinis intended to include a portion, e.g., a domain/motif, of an IMIprotein that, when produced in a plant increases the plant's resistanceto an imidazolinone herbicide as compared to a wild-type variety of theplant. Methods for quantitating increased resistance to imidazolinoneherbicides are provided in the Examples below. Biologically activeportions of an IMI protein include peptides comprising SEQ ID NO:2, SEQID NO:4 or SEQ ID NO:6 which include fewer amino acids than a fulllength IMI protein and impart increased resistance to an imidazolinoneherbicide upon expression in a plant. Typically, biologically activeportions (e.g., peptides which are, for example, 5, 10, 15, 20, 30, 35,36, 37, 38, 39, 40, 50, 100 or more amino acids in length) comprise adomain or motif with at least one activity of an IMI protein. Moreover,other biologically active portions in which other regions of thepolypeptide are deleted, can be prepared by recombinant techniques andevaluated for one or more of the activities described herein.Preferably, the biologically active portions of an IMI protein includeone or more conserved domains selected from the group consisting of aDomain A, a Domain B, a Domain C, a Domain D and a Domain E, wherein theconserved domain contains a mutation.

The invention also provides IMI chimeric or fusion polypeptides. As usedherein, an IMI “chimeric polypeptide” or “fusion polypeptide” comprisesan IMI polypeptide operably linked to a non-IMI polypeptide. A “non-IMIpolypeptide” refers to a polypeptide having an amino acid sequence thatis not substantially identical to an IMI polypeptide, e.g., apolypeptide that is not an IMI isoenzyme, which peptide performs adifferent function than an IMI polypeptide. Within the fusionpolypeptide, the term “operably linked” is intended to indicate that theIMI polypeptide and the non-IMI polypeptide are fused to each other sothat both sequences fulfill the proposed function attributed to thesequence used. The non-IMI polypeptide can be fused to the N-terminus orC-terminus of the IMI polypeptide. For example, in one embodiment, thefusion polypeptide is a GST-IMI fusion polypeptide in which the IMIsequence is fused to the C-terminus of the GST sequence. Such fusionpolypeptides can facilitate the purification of recombinant IMIpolypeptides. In another embodiment, the fusion polypeptide is an IMIpolypeptide containing a heterologous signal sequence at its N-terminus.In certain host cells (e.g., mammalian host cells), expression and/orsecretion of an IMI polypeptide can be increased through use of aheterologous signal sequence.

An isolated nucleic acid molecule encoding an IMI polypeptide havingsequence identity to a polypeptide encoded by a polynucleotide sequenceof SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 can be created by introducingone or more nucleotide substitutions, additions or deletions into anucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 such thatone or more amino acid substitutions, additions or deletions areintroduced into the encoded polypeptide. Mutations can be introducedinto a sequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in an IMI polypeptide ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of an IMI coding sequence, such asby saturation mutagenesis, and the resultant mutants can be screened foran IMI activity described herein to identify mutants that retain IMIactivity. Following mutagenesis of the sequence of SEQ ID NO:1, SEQ IDNO:3 or SEQ ID NO:5, the encoded polypeptide can be expressedrecombinantly and the activity of the polypeptide can be determined byanalyzing the imidazolinone resistance of a plant expressing thepolypeptide as described in the Examples below.

To determine the percent sequence identity of two amino acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gaps 19can be introduced in the sequence of one polypeptide for optimalalignment with the other polypeptide). The amino acid residues atcorresponding amino acid positions are then compared. When a position inone sequence is occupied by the same amino acid residue as thecorresponding position in the other sequence, then the molecules areidentical at that position. The same type of comparison can be madebetween two nucleic acid sequences. The percent sequence identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (i.e., percent sequenceidentity=numbers of identical positions/total numbers of positions×100).For the purposes of the invention, the percent sequence identity,between two nucleic acid or polypeptide sequences is determined usingthe Vector NTI 6.0 (PC) software package (InforMax, 7600 Wisconsin Ave.,Bethesda, Md. 20814). A gap opening penalty of 15 and a gap extensionpenalty of 6.66 are used for determining the percent identity of twonucleic acids. A gap opening penalty of 10 and a gap extension penaltyof 0.1 are used for determining the percent identity of twopolypeptides. All other parameters are set at the default settings.

It is to be understood that for the purposes of determining sequenceidentity, when comparing a DNA sequence to an RNA sequence, a thymidinenucleotide is equivalent to a uracil nucleotide. Preferably, theisolated IMI polypeptides included in the present invention are at leastabout 50-60%, preferably at least about 60-70%, and more preferably atleast about 70-75%, 75-80%, 80-85%, 85-90% or 90-95%, and mostpreferably at least about 96%, 97%, 98%, 99% or more identical to anentire amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ IDNO:6. In another embodiment, the isolated IMI polypeptides included inthe present invention are at least about 50-60%, preferably at leastabout 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%,85-90% or 90-95%, and most preferably at least about 96%, 97%, 98%, 99%or more identical to an entire amino acid sequence shown in SEQ ID NO:2,SEQ ID NO:4 or SEQ ID NO:6. Additionally, optimized IMI nucleic acidscan be created. Preferably, an optimized IMI nucleic acid encodes an IMIpolypeptide that modulates a plant's tolerance to imidazolinoneherbicides, and more preferably increases a plant's tolerance to animidazolinone herbicide upon its over-expression in the plant. As usedherein, “optimized” refers to a nucleic acid that is geneticallyengineered to increase its expression in a given plant or animal. Toprovide plant optimized IMI nucleic acids, the DNA sequence of the genecan be modified to 1) comprise codons preferred by highly expressedplant genes; 2) comprise an A+T content in nucleotide base compositionto that substantially found in plants; 3) form a plant initiationsequence, 4) eliminate sequences that cause destabilization,inappropriate polyadenylation, degradation and termination of RNA, orthat form secondary structure hairpins or RNA splice sites. Increasedexpression of IMI nucleic acids in plants can be achieved by utilizingthe distribution frequency of codon usage in plants in general or aparticular plant. Methods for optimizing nucleic acid expression inplants can be found in EPA 0359472; EPA 0385962; PCT Application No. WO91/16432; U.S. Pat. Nos. 5,380,831; 5,436,391; Perlack et at., 1991Proc. Natl. Acad. Sci. USA 88:3324-3328; and Murray et at., 1989 NucleicAcids Res. 17:477-498.

As used herein, “frequency of preferred codon usage” refers to thepreference exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. To determine the frequency ofusage of a particular codon in a gene, the number of occurrences of thatcodon in the gene is divided by the total number of occurrences of allcodons specifying the same amino acid in the gene. Similarly, thefrequency of preferred codon usage exhibited by a host cell can becalculated by averaging frequency of preferred codon usage in a largenumber of genes expressed by the host cell. It is preferable that thisanalysis be limited to genes that are highly expressed by the host cell.The percent deviation of the frequency of preferred codon usage for asynthetic gene from that employed by a host cell is calculated first bydetermining the percent deviation of the frequency of usage of a singlecodon from that of the host cell followed by obtaining the averagedeviation over all codons. As defined herein, this calculation includesunique codons (i.e., ATG and TGG). In general terms, the overall averagedeviation of the codon usage of an optimized gene from that of a hostcell is calculated using the equation 1A=n=1 Z X_(n)−Y_(n) X_(n) times100 Z where X_(n)=frequency of usage for codon n in the host cell;Y_(n)=frequency of usage for codon n in the synthetic gene, n representsan individual codon that specifies an amino acid and the total number ofcodons is Z. The overall deviation of the frequency of codon usage, A,for all amino acids should preferably be less than about 25%, and morepreferably less than about 10%.

Hence, an IMI nucleic acid can be optimized such that its distributionfrequency of codon usage deviates, preferably, no more than 25% fromthat of highly expressed plant genes and, more preferably, no more thanabout 10%. In addition, consideration is given to the percentage G+Ccontent of the degenerate third base (monocotyledons appear to favor G+Cin this position, whereas dicotyledons do not). It is also recognizedthat the XCG (where X is A, T, C, or G) nucleotide is the leastpreferred codon in dicots whereas the XTA codon is avoided in bothmonocots and dicots. Optimized IMI nucleic acids of this invention alsopreferably have CG and TA doublet avoidance indices closelyapproximating those of the chosen host plant (i.e., Triticum aestivum).More preferably these indices deviate from that of the host by no morethan about 10-15%.

In addition to the nucleic acid molecules encoding the IMI polypeptidesdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules that are antisense thereto. Antisensepolynucleotides are thought to inhibit gene expression of a targetpolynucleotide by specifically binding the target polynucleotide andinterfering with transcription, splicing, transport, translation and/orstability of the target polynucleotide. Methods are described in theprior art for targeting the antisense polynucleotide to the chromosomalDNA, to a primary RNA transcript or to a processed mRNA. Preferably, thetarget regions include splice sites, translation initiation codons,translation termination codons, and other sequences within the openreading frame.

The term “antisense”, for the purposes of the invention, refers to anucleic acid comprising a polynucleotide that is sufficientlycomplementary to all or a portion of a gene, primary transcript orprocessed mRNA, so as to interfere with expression of the endogenousgene. “Complementary” polynucleotides are those that are capable of basepairing according to the standard Watson-Crick complementarity rules.Specifically, purines will base pair with pyrimidines to form acombination of guanine paired with cytosine (G:C) and adenine pairedwith either thymine (A:T) in the case of DNA, or adenine paired withuracil (A:U) in the case of RNA. It is understood that twopolynucleotides may hybridize to each other even if they are notcompletely complementary to each other, provided that each has at leastone region that is substantially complementary to the other. The term“antisense nucleic acid” includes single stranded RNA as well asdouble-stranded DNA expression cassettes that can be transcribed toproduce an antisense RNA. “Active” antisense nucleic acids are antisenseRNA molecules that are capable of selectively hybridizing with a primarytranscript or mRNA encoding a polypeptide having at least 80% sequenceidentity with the polypeptide sequence of SEQ ID NO:2, SEQ ID NO:4 orSEQ ID NO:6.

In addition to the IMI nucleic acids and polypeptides described above,the present invention encompasses these nucleic acids and polypeptidesattached to a moiety. These moieties include, but are not limited to,detection moieties, hybridization moieties, purification moieties,delivery moieties, reaction moieties, binding moieties, and the like. Atypical group of nucleic acids having moieties attached are probes andprimers. Probes and primers typically comprise a substantially isolatedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, preferably about 25, more preferably about 40, 50 or 75consecutive nucleotides of a sense strand of the sequence set forth inSEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, an anti-sense sequence of thesequence set forth in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5, ornaturally occurring mutants thereof. Primers based on a nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 can be used in PCRreactions to clone IMI homologs. Probes based on the IMI nucleotidesequences can be used to detect transcripts or genomic sequencesencoding the same or homologous polypeptides. In preferred embodiments,the probe further comprises a label group attached thereto, e.g. thelabel group can be a radioisotope, a fluorescent compound, an enzyme, oran enzyme co-factor. Such probes can be used as a part of a genomicmarker test kit for identifying cells which express an IMI polypeptide,such as by measuring a level of an IMI-encoding nucleic acid, in asample of cells, e.g., detecting IMI mRNA levels or determining whethera genomic IMI gene has been mutated or deleted.

The invention further provides an isolated recombinant expression vectorcomprising an IMI nucleic acid as described above, wherein expression ofthe vector in a host cell results in increased resistance to animidazolinone herbicide as compared to a wild type variety of the hostcell. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operably linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel, Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990) or see:Gruber and Crosby, in: Methods in Plant Molecular Biology andBiotechnology, eds. Glick and Thompson, Chapter 7, 89-108, CRC Press:Boca Raton, Fla., including the references therein. Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells and those that direct expression ofthe nucleotide sequence only in certain host cells or under certainconditions. It will be appreciated by those skilled in the art that thedesign of the expression vector can depend on such factors as the choiceof the host cell to be transformed, the level of expression ofpolypeptide desired, etc. The expression vectors of the invention can beintroduced into host cells to thereby produce polypeptides or peptides,including fusion polypeptides or peptides, encoded by nucleic acids asdescribed herein (e.g., IMI polypeptides, fusion polypeptides, etc.).

In a preferred embodiment of the present invention, the IMI polypeptidesare expressed in plants and plants cells such as unicellular plant cells(such as algae) (see Falciatore et al., 1999 Marine Biotechnology1(3):239-251 and references therein) and plant cells from higher plants(e.g., the spermatophytes, such as crop plants). An IMI polynucleotidemay be “introduced” into a plant cell by any means, includingtransfection, transformation or transduction, electroporation, particlebombardment, agroinfection, biolistics and the like.

Suitable methods for transforming or transfecting host cells includingplant cells can be found in Sambrook, et al. (Molecular Cloning: ALaboratory Manual. 2^(nd), ed., Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) andother laboratory manuals such as Methods in Molecular Biology, 1995,Vol. 44, Agrobacterium protocols, ed: Gartland and Davey, Humana Press,Totowa, N.J. As increased resistance to imidazolinone herbicides is ageneral trait wished to be inherited into a wide variety of plants likemaize, wheat, rye, oat, triticale, rice, barley, soybean, peanut,cotton, rapeseed and canola, manihot, pepper, sunflower and tagetes,solanaceous plants like potato, tobacco, eggplant, and tomato, Viciaspecies, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species,trees (oil palm, coconut), perennial grasses and forage crops, thesecrop plants are also preferred target plants for a genetic engineeringas one further embodiment of the present invention. In a preferredembodiment, the plant is a wheat plant. Forage crops include, but arenot limited to, Wheatgrass, Canarygrass, Bromegrass, Wildrye Grass,Bluegrass, Orchardgrass, Alfalfa, Salfoin, Birdsfoot Trefoil, AlsikeClover, Red Clover and Sweet Clover.

In one embodiment of the present invention, transfection of an IMIpolynucleotide into a plant is achieved by Agrobacterium mediated genetransfer. One transformation method known to those of skill in the artis the dipping of a flowering plant into an Agrobacteria solution,wherein the Agrobacteria contains the IMI nucleic acid, followed bybreeding of the transformed gametes. Agrobacterium mediated planttransformation can be performed using for example the GV3101(pMP90)(Koncz and Schell, 1986 Mol. Gen. Genet. 204:383-396) or LBA4404(Clontech) Agrobacterium tumefaciens strain. Transformation can beperformed by standard transformation and regeneration techniques(Deblaere et al., 1994 Nucl. Acids. Res. 13:4777-4788; Gelvin, StantonB. and Schilperoort, Robert A, Plant Molecular Biology Manual, 2^(nd)Ed.—Dordrecht: Kluwer Academic Publ., 1995.—in Sect., Ringbuc ZentraleSignatur: BTI 1-P ISBN 0-7923-2731-4; Glick, Bernard R. and Thompson,John E., Methods in Plant Molecular Biology and Biotechnology, BocaRaton: CRC Press, 1993 360 S., ISBN 0-8493-5164-2). For example,rapeseed can be transformed via cotyledon or hypocotyl transformation(Moloney et al., 1989 Plant cell Report 8:238-242; De Block et al., 1989Plant Physiol. 91:694-701). Use of antibiotica for Agrobacterium andplant selection depends on the binary vector and the Agrobacteriumstrain used for transformation. Rapeseed selection is normally performedusing kanamycin as selectable plant marker. Agrobacterium mediated genetransfer to flax can be performed using, for example, a techniquedescribed by Mlynarova et al., 1994 Plant Cell Report 13:282-285.Additionally, transformation of soybean can be performed using forexample a technique described in European Patent No. 0424 047, U.S. Pat.No. 5,322,783, European Patent No. 0397 687, U.S. Pat. Nos. 5,376,543 or5,169,770. Transformation of maize can be achieved by particlebombardment, polyethylene glycol mediated DNA uptake or via the siliconcarbide fiber technique. (See, for example, Freeling and Walbot “Themaize handbook” Springer Verlag: New York (1993) ISBN 3-540-97826-7). Aspecific example of maize transformation is found in U.S. Pat. No.5,990,387 and a specific example of wheat transformation can be found inPCT Application No. WO 93/07256.

According to the present invention, the introduced IMI polynucleotidemay be maintained in the plant cell stably if it is incorporated into anon-chromosomal autonomous replicon or integrated into the plantchromosomes. Alternatively, the introduced IMI polynucleotide may bepresent on an extra-chromosomal non-replicating vector and betransiently expressed or transiently active. In one embodiment, ahomologous recombinant microorganism can be created wherein the IMIpolynucleotide is integrated into a chromosome, a vector is preparedwhich contains at least a portion of an AHAS gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the endogenous AHAS gene and to create an IMIgene. To create a point mutation via homologous recombination, DNA-RNAhybrids can be used in a technique known as chimeraplasty (Cole-Strausset al., 1999 Nucleic Acids Research 27(5):1323-1330 and Kmiec, 1999 Genetherapy American Scientist 87(3):240-247). Other homologousrecombination procedures in Triticum species are also well known in theart and are contemplated for use herein.

In the homologous recombination vector, the IMI gene can be flanked atits 5′ and 3′ ends by an additional nucleic acid molecule of the AHASgene to allow for homologous recombination to occur between theexogenous IMI gene carried by the vector and an endogenous AHAS gene, ina microorganism or plant. The additional flanking AHAS nucleic acidmolecule is of sufficient length for successful homologous recombinationwith the endogenous gene. Typically, several hundreds of base pairs upto kilobases of flanking DNA (both at the 5′ and 3′ ends) are includedin the vector (see e.g., Thomas, K. R., and Capecchi, M. R., 1987 Cell51:503 for a description of homologous recombination vectors or Streppet al., 1998 PNAS, 95(8):4368-4373 for cDNA based recombination inPhyscomitrella patens). However, since the IMI gene normally differsfrom the AHAS gene at very few amino acids, a flanking sequence is notalways necessary. The homologous recombination vector is introduced intoa microorganism or plant cell (e.g., via polyethylene glycol mediatedDNA), and cells in which the introduced IMI gene has homologouslyrecombined with the endogenous AHAS gene are selected using art-knowntechniques.

In another embodiment, recombinant microorganisms can be produced thatcontain selected systems that allow for regulated expression of theintroduced gene. For example, inclusion of an IMI gene on a vectorplacing it under control of the lac operon permits expression of the IMIgene only in the presence of IPTG. Such regulatory systems are wellknown in the art.

Whether present in an extra-chromosomal non-replicating vector or avector that is integrated into a chromosome, the IMI polpucleotidepreferably resides in a plant expression cassette. A plant expressioncassette preferably contains regulatory sequences capable of drivinggene expression in plant cells that are operably linked so that eachsequence can fulfill its function, for example, termination oftranscription by polyadenylation signals. Preferred polyadenylationsignals are those originating from Agrobacterium tumefaciens t-DNA suchas the gene 3 known as octopine synthase of the Ti-plasmid pTiACH5(Gielen et al., 1984 EMBO J. 3:835) or functional equivalents thereof,but also all other terminators functionally active in plants aresuitable. As plant gene expression is very often not limited ontranscriptional levels, a plant expression cassette preferably containsother operably linked sequences like translational enhancers such as theoverdrive-sequence containing the 5′-untranslated leader sequence fromtobacco mosaic virus enhancing the polypeptide per RNA ratio (Gallie etal., 1987 Nucl. Acids Research 15:8693-8711). Examples of plantexpression vectors include those detailed in: Becker, D. et al., 1992New plant binary vectors with selectable markers located proximal to theleft border, Plant Mol. Biol. 20:1195-1197; Bevan, M. W., 1984 BinaryAgrobacterium vectors for plant transformation, Nucl. Acid. Res.12:8711-8721; and Vectors for Gene Transfer in Higher Plants; in:Transgenic Plants, Vol. 1, Engineering and Utilization, eds.: Kung andR. Wu, Academic Press, 1993, S. 15-38.

Plant gene expression should be operably linked to an appropriatepromoter conferring gene expression in a timely, cell or tissue specificmanner. Promoters useful in the expression cassettes of the inventioninclude any promoter that is capable of initiating transcription in aplant cell. Such promoters include, but are not limited to those thatcan be obtained from plants, plant viruses and bacteria that containgenes that are expressed in plants, such as Agrobacterium and Rhizobium.

The promoter may be constitutive, inducible, developmentalstage-preferred, cell type-preferred, tissue-preferred ororgan-preferred. Constitutive promoters are active under mostconditions. Examples of constitutive promoters include the CaMV 19S and35S promoters (Odell et al. 1985 Nature 313:810-812), the sX CaMV 35Spromoter (Kay et al. 1987 Science 236:1299-1302) the Sep 1 promoter, therice actin promoter (McElroy et al. 1990 Plant Cell 2:163-171), theArabidopsis actin promoter, the ubiquitan promoter (Christensen et al.1989 Plant Molec Biol. 18:675-689); pEmu (Last et al. 1991 Theor ApplGenet. 81:581-588), the figwort mosaic virus 35S promoter, the Smaspromoter (Velten et al. 1984 EMBO J. 3:2723-2730), the GRP1-8 promoter,the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439),promoters from the T-DNA of Agrobacterium, such as mannopine synthase,nopaline synthase, and octopine synthase, the small subunit of ribulosebiphosphate carboxylase (ssuRUBISCO) promoter, and the like.

Inducible promoters are active under certain environmental conditions,such as the presence or absence of a nutrient or metabolite, heat orcold, light, pathogen attack, anaerobic conditions, and the like. Forexample, the hsp80 promoter from Brassica is induced by heat shock, thePPDK promoter is induced by light, the PR-1 promoter from tobacco,Arabidopsis and maize are inducible by infection with a pathogen, andthe Adh1 promoter is induced by hypoxia and cold stress. Plant geneexpression can also be facilitated via an inducible promoter (for reviewsee Gatz, 1997 Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108).Chemically inducible promoters are especially suitable if time-specificgene expression is desired. Examples of such promoters are a salicylicacid inducible promoter (PCT Application No. WO 95/19443), atetracycline inducible promoter (Gatz et al., 1992 Plant J. 2:397-404)and an ethanol inducible promoter (PCT Application No. WO 93/21334).

Developmental stage-preferred promoters are preferentially expressed atcertain stages of development. Tissue and organ preferred promotersinclude those that are preferentially expressed in certain tissues ororgans, such as leaves, roots, seeds, or xylem. Examples of tissuepreferred and organ preferred promoters include, but are not limited tofruit-preferred, ovule-preferred, male tissue-preferred, seed-preferred,integument-preferred, tuber-preferred, stalk-preferred,pericarp-preferred, and leaf-preferred, stigma-preferred,pollen-preferred, anther-preferred, a petal-preferred, sepal-preferred,pedicel-preferred, silique-preferred, stem-preferred, root-preferredpromoters and the like. Seed preferred promoters are preferentiallyexpressed during seed development and/or germination. For example, seedpreferred promoters can be embryo-preferred, endosperm preferred andseed coat-preferred. See Thompson et al., 1989 BioEssays 10:108.Examples of seed preferred promoters include, but are not limited tocellulose synthase (cel A), Cim1, gamma-zein, globulin-1, maize 19 kDzein (cZ19B1) and the like.

Other suitable tissue-preferred or organ-preferred promoters include thenapin-gene promoter from rapeseed (U.S. Pat. No 5,608,152), theUSP-promoter from Vicia faba (Baeumlein et al., 1991 Mot Gen Genet.225(3):459-67), the oleosin-promoter from Arabidopsis (PCT ApplicationNo. WO 98/45461), the phaseolin-promoter from Phaseolus vulgaris (U.S.Pat. No. 5,504,200), the Bce4-promoter from Brassica (PCT ApplicationNo. WO 91/13980) or the legumin B4 promoter (LeB4; Baeumlein et al.,1992 Plant Journal, 2(2):233-9) as well as promoters conferring seedspecific expression in monocot plants like maize, barley, wheat, rye,rice, etc. Suitable promoters to note are the 1pt2 or 1pt1-gene promoterfrom barley (PCT Application No. WO 95/15389 and PCT Application No. WO95/23230) or those described in PCT Application No. WO 99/16890(promoters from the barley hordein-gene, rice glutelin gene, rice oryzingene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, oatglutelin gene, Sorghum kasirin-gene and rye secalin gene).

Other promoters useful in the expression cassettes of the inventioninclude, but are not limited to, the major chlorophyll a/b bindingprotein promoter, histone promoters, the Ap3 promoter, the conglycinpromoter, the napin promoter, the soy bean lectin promoter, the maize 15kD zein promoter, the 22 kD zein promoter, the 27 KD zein promoter, theg-zein promoter, the waxy, shrunken 1, shrunken 2 and bronze promoters,the Zm13 promoter (U.S. Pat. No. 5,086,169), the maize polygalacturonasepromoters (PG) (U.S. Pat. Nos. 5,412,085 and 5,545,546) and the SGB6promoter (U.S. Pat. No. 5,470,359), as well as synthetic or othernatural promoters.

Additional flexibility in controlling heterologous gene expression inplants may be obtained by using DNA binding domains and responseelements from heterologous sources (i.e., DNA binding domains fromnon-plant sources). An example of such a heterologous DNA binding domainis the LexA DNA binding domain (Brent and Ptashne, 1985 Cell43:729-736).

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but they also apply to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein. A host cell can be any prokaryotic or eukaryotic cell. Forexample, an IMI polynucleotide can be expressed in bacterial cells suchas C. glutamicum, insect cells, fungal cells or mammalian cells (such asChinese hamster ovary cells (CHO) or COS cells), algae, ciliates, plantcells, fungi or other microorganisms like C. glutamicum. Other suitablehost cells are known to those skilled in the art.

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) an IMIpolynucleotide. Accordingly, the invention further provides methods forproducing IMI polypeptides using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of invention(into which a recombinant expression vector encoding an IMI polypeptidehas been introduced, or into which genome has been introduced a geneencoding a wild-type or IMI polypeptide) in a suitable medium until IMIpolypeptide is produced. In another embodiment, the method furthercomprises isolating IMI polypeptides from the medium or the host cell.Another aspect of the invention pertains to isolated IMI polypeptides,and biologically active portions thereof. An “isolated” or “purified”polypeptide or biologically active portion thereof is free of some ofthe cellular material when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized. Thelanguage “substantially free of cellular material” includes preparationsof IMI polypeptide in which the polypeptide is separated from some ofthe cellular components of the cells in which it is naturally orrecombinantly produced. In one embodiment, the language “substantiallyfree of cellular material” includes preparations of an IMI polypeptidehaving less than about 30% (by dry weight) of non-IMI material (alsoreferred to herein as a “contaminating polypeptide”), more preferablyless than about 20% of non-IMI material, still more preferably less thanabout 10% of non-IMI material, and most preferably less than about 5%non-IMI material.

When the IMI polypeptide, or biologically active portion thereof, isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the polypeptide preparation. The language“substantially free of chemical precursors or other chemicals” includespreparations of IMI polypeptide in which the polypeptide is separatedfrom chemical precursors or other chemicals that are involved in thesynthesis of the polypeptide. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of an IMI polypeptide having less than about 30% (by dryweight) of chemical precursors or non-IMI chemicals, more preferablyless than about 20% chemical precursors or non-IMI chemicals, still morepreferably less than about 10% chemical precursors or non-IMI chemicals,and most preferably less than about 5% chemical precursors or non-IMIchemicals. In preferred embodiments, isolated polypeptides, orbiologically active portions thereof, lack contaminating polypeptidesfrom the same organism from which the IMI polypeptide is derived.Typically, such polypeptides are produced by recombinant expression of,for example, a Triticum aestivum IMI polypeptide in plants other thanTriticum aestivum or microorganisms such as C. glutamicum, ciliates,algae or fungi.

The IMI polynucleotide and polypeptide sequences of the invention have avariety of uses. The nucleic acid and amino acid sequences of thepresent invention can be used to transform plants, thereby modulatingthe plant's resistance to imidazolinone herbicides. Accordingly, theinvention provides a method of producing a transgenic plant havingincreased tolerance to an imidazolinone herbicide comprising, (a)transforming a plant cell with one or more expression vectors comprisingone or more IMI nucleic acids, and (b) generating from the plant cell atransgenic plant with an increased resistance to an imidazolinoneherbicide as compared to a wild type variety of the plant. In oneembodiment, the multiple IMI nucleic acids are derived from differentgenomes. Also included in the present invention are methods of producinga transgenic plant having increased tolerance to an imidazolinoneherbicide comprising, (a) transforming a plant cell with an expressionvector comprising an IMI nucleic acid, wherein the nucleic acid is anon-Imi1 nucleic acid and (b) generating from the plant cell atransgenic plant with an increased resistance to an imidazolinoneherbicide as compared to a wild type variety of the plant.

The present invention includes methods of modifying a plant's toleranceto an imidazolinone herbicide comprising modifying the expression of oneor more IMI nucleic acids. Preferably, the nucleic acids are located onor derived from different genomes. The plant's resistance to theimidazolinone herbicide can be increased or decreased as achieved byincreasing or decreasing the expression of an IMI polynucleotide,respectively. Preferably, the plant's resistance to the imidazolinoneherbicide is increased by increasing expression of an IMIpolynucleotide. Expression of an IMI polynucleotide can be modified byany method known to those of skill in the art. The methods of increasingexpression of IMI polynucleotides can be used wherein the plant iseither transgenic or not transgenic. In cases when the plant istransgenic, the plant can be transformed with a vector containing any ofthe above described IMI coding nucleic acids, or the plant can betransformed with a promoter that directs expression of endogenous IMIpolynucleotides in the plant, for example. The invention provides thatsuch a promoter can be tissue specific or developmentally regulated.Alternatively, non-transgenic plants can have endogenous IMIpolynucleotide expression modified by inducing a native promoter. Theexpression of polynucleotides comprising SEQ ID NO:1, SEQ ID NO:3 or SEQID NO:5 in target plants can be accomplished by, but is not limited to,one of the following examples: (a) constitutive promoter, (b)chemical-induced promoter, and (c) engineered promoter over-expressionwith for example zinc-finger derived transcription factors (Greisman andPabo, 1997 Science 275:657).

In a preferred embodiment, transcription of the IMI polynucleotide ismodulated using zinc-finger derived transcription factors (ZFPs) asdescribed in Greisman and Pabo, 1997 Science 275:657 and manufactured bySangamo Biosciences, Inc. These ZFPs comprise both a DNA recognitiondomain and a functional domain that causes activation or repression of atarget nucleic acid such as an IMI nucleic acid. Therefore, activatingand repressing ZFPs can be created that specifically recognize the IMIpolynucleotide promoters described above and used to increase ordecrease IMI polynucleotide expression in a plant, thereby modulatingthe herbicide resistance of the plant.

As described in more detail above, the plants produced by the methods ofthe present invention can be monocots or dicots. The plants can beselected from maize, wheat, rye, oat, triticale, rice, barley, soybean,peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, tagetes,solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species,pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut,perennial grass and forage crops, for example. In a preferredembodiment, the plant is a wheat plant. Forage crops include, but arenot limited to, Wheatgrass, Canarygrass, Bromegrass, Wildrye Grass,Bluegrass, Orchardgrass, Alfalfa, Salfoin, Birdsfoot Trefoil, AlsikeClover, Red Clover and Sweet Clover. In a preferred embodiment, theplant is a wheat plant. In each of the methods described above, theplant cell includes, but is not limited to, a protoplast, gameteproducing cell, and a cell that regenerates into a whole plant. As usedherein, the term “transgenic” refers to any plant, plant cell, callus,plant tissue or plant part, that contains all or part of at least onerecombinant polynucleotide. In many cases, all or part of therecombinant polynucleotide is stably integrated into a chromosome orstable extra-chromosomal element, so that it is passed on to successivegenerations.

As described above, the present invention teaches compositions andmethods for increasing the imidazolinone resistance of a wheat plant orseed as compared to a wild-type variety of the plant or seed. In apreferred embodiment, the imidazolinone resistance of a wheat plant orseed is increased such that the plant or seed can withstand animidazolinone herbicide application of preferably approximately 10-400 gai ha⁻¹, more preferably 20-160 g ai ha⁻¹, and most preferably 40-80 gai ha⁻¹. As used herein, to “withstand” an imidazolinone herbicideapplication means that the plant is either not killed or not injured bysuch application.

Additionally provided herein is a method of controlling weeds within thevicinity of a wheat plant, comprising applying an imidazolinoneherbicide to the weeds and to the wheat plant, wherein the wheat planthas increased resistance to the imidazolinone herbicide as compared to awild type variety of the wheat plant, and wherein the plant comprisesone or more IMI nucleic acids. In one embodiment, the plant comprisesmultiple IMI nucleic acids located on or derived from different genomes.In another embodiment, the plant comprises a non-Imi1 nucleic acid. Byproviding for wheat plants having increased resistance to imidazolinone,a wide variety of formulations can be employed for protecting wheatplants from weeds, so as to enhance plant growth and reduce competitionfor nutrients. An imidazolinone herbicide can be used by itself forpre-emergence, post-emergence, pre-planting and at-planting control ofweeds in areas surrounding the wheat plants described herein or animidazolinone herbicide formulation can be used that contains otheradditives. The imidazolinone herbicide can also be used as a seedtreatment. Additives found in an imidazolinone herbicide formulationinclude other herbicides, detergents, adjuvants, spreading agents,sticking agents, stabilizing agents, or the like. The imidazolinoneherbicide formulation can be a wet or dry preparation and can include,but is not limited to, flowable powders, emulsifiable concentrates andliquid concentrates. The imidazolinone herbicide and herbicideformulations can be applied in accordance with conventional methods, forexample, by spraying, irrigation, dusting, or the like.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art to which this invention pertains.

It should also be understood that the foregoing relates to preferredembodiments of the present invention and that numerous changes may bemade therein without departing from the scope of the invention. Theinvention is further illustrated by the following examples, which arenot to be construed in any way as imposing limitations upon the scopethereof. On the contrary, it is to be clearly understood that resort maybe had to various other embodiments, modifications, and equivalentsthereof, which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the present invention and/or the scope of the appended claims.

EXAMPLES Example 1

Mutagenesis and Selection of Resistant Gunner Wheat Lines

The imidazolinone resistant wheat was derived through mutation andconventional selection and breeding. Initial seed mutagenesis wasconducted as follows:

1. Seeds of the hard red spring wheat variety Gunner, were pre-soaked intap water.

2. After decanting the tap water, a solution of 0.03% Ethylmethanesulfonate (EMS) and 0.02% Diethyl Sulfate (DES) was poured in to theseed container. The container was shaken every 10-15 minutes during thecourse of a two-hour treatment.

3. The EMS and DES solution was decanted and a solution of 0.02% sodiumazide in 0.001M phosphate buffer was added.

4. Step three was repeated.

5. Seeds were then rinsed in tap water and dried. After drying, seedswere planted.

6. Planted seeds represented the M1 generation. Seed harvested from M1plants represented the M2 generation.

M2 seed were planted and emerged plants were treated with imazamoxherbicide at approximately the 2-3 leaf stage at rates which would killsusceptible wheat. A total of nine herbicide tolerant plants wereselected and re-planted in a greenhouse. Progeny seed were collectedfrom each of the nine plants. This seed was planted in a greenhouse.Plants were sprayed with imazamox herbicide at 80 g. a.i./ha.+1.0%Sun-It adjuvant (v/v) and evaluated for tolerance. A total of twelveplants of two lines, designated HRS198205 and HRS198208, were identifiedas most tolerant. Segregation for herbicide tolerance in each line wasconsistent with a single semi-dominant gene. Progeny seed of the 24plants were collected and re-planted in a greenhouse for furtherevaluation and selection. Plants were sprayed with imazamox herbicide at80 g. a.i./ha.+1.0% Sun-It adjuvant (v/v) resulting in theidentification of sub-lines that exhibited the highest level oftolerance. In addition, the sub-lines were determined to have thephenotypic characteristics of Gunner.

Progeny seed of HRS 198205 and HRS 198208 were collected and planted inthe field. Field plots were sprayed with imazamox herbicide at 80 g.a.i./ha.+1.0% Sun-It adjuvant (v/v). All plants in each line exhibitedthe same level of acceptable tolerance to imazamox herbicide. Based uponthese results, seed harvested from plots of five of the HRS 198205sub-lines were combined into a single lot that was designated AP205CL(referred to above as Gunner IMI 205). Additionally, seed harvested fromplots of five of the HRS198208 sub-lines were combined into a single lotthat was designated AP602CL (referred to above as Gunner IMI 208). Seedincreases were conducted at several locations. All seed increases weresprayed with imazamox herbicide at 40 g. a.i./ha+0.25% (v/v) non-ionicsurfactant. No herbicide susceptible plants were observed. In addition,all plants were comparable to plants of the variety Gunner.

Example 2

Mutagenesis and Selection of Resistant Madsen Wheat Lines

Seeds of the soft white winter wheat variety Madsen, were pre-soaked intap water. After decanting the tap water, a solution of 0.03% EMS and0.02% DES was poured into the seed container. The container was shakenevery 10-15 minutes during the course of a two-hour treatment. The EMSand DES solution was decanted and a solution of 0.02% sodium azide in0.001M phosphate buffer was added. The container again was shaken every10-15 minutes during the course of a two-hour treatment. Seeds were thenrinsed in tap water and dried. After drying, seeds were planted. Plantedseeds represented the M₁ generation. M₁ plants were allowed toself-pollinate and the M₂ seed harvested as a bulk. Approximately 0.2hectares of M₂ seed were planted in the field and resultant plantstreated with an imazamox rate of 40 g. ai/ha. Twelve M₂ plants wereidentified as tolerant. These plants were dug, and sent to Pullman,Wash. for vernalization and M₃ seed production. M3 seed from each of theM₂ plants were planted in the greenhouse and resultant plants werevernalized for 8 weeks, then treated with 80 g. ai/ha of Imazamox.Plants were selected based upon observed levels of tolerance andrepotted for seed production. The M_(2:3) line designated Madsen1selected as tolerant to 40 g/ha imazamox as an M₂ was confirmed astolerant to imazamox at the 80 g/ha rate applied to M₃ progeny.Subsequent molecular characterization determined that Madsen1 had amutation in the Als2 AHAS gene known to confer tolerance toimidazolinone herbicides.

Example 3

Tolerance of the AP205CL and AP602CL Wheat Plants to ImidazolinoneHerbicides

Both the AP205CL and AP602CL wheat plants are tolerant to imidazolinoneherbicides due to a mutation of the AHAS enzyme that is resistant toinhibition by these herbicides in vitro. This is demonstrated bycomparison of the activity of the AHAS enzyme extracted from wild typewheat to the AHAS activity extracted from herbicide tolerant AP205CLplants (FIG. 2A) and AP602CL plants (FIG. 2B). The values in FIG. 1 areexpressed as a percent of uninhibited activity. The AHAS enzyme fromwild type Gunner wheat exhibits a 1 to 35 percent reduction of activityin the presence of a low concentration (1 M) of imidazolinone herbicideimazamox. This activity continues to decline to nearly 100 percentinhibition of the enzyme at higher herbicide concentrations (100 M). Incontrast, the AHAS enzyme extracted from herbicide tolerant AP205CLplants retains nearly 90 percent of its activity at the 1 M imazamoxconcentration, and approximately one third of its activity at the higher(50 M to 100 M) concentrations. The AHAS enzyme extracted from herbicidetolerant AP602CL plants retains nearly 80-100 percent of its activity atthe 1 M imazamox concentration, and nearly half of its activity at thehigher (50 M to 100 M) concentrations. These levels of activity aresufficient to allow the tolerant wheat plants to survive the applicationof imazamox, as was observed during the selection process (Example 1).

Example 3

Tolerance of the Madsen Wheat Plants to Imidazolinone Herbicides

Madsen1 was evaluated for tolerance to the imidazolinone herbicideimazamox at 40 and 80 g/ha in a greenhouse trial. The susceptible wheatcultivar Tea1 was used as a control. Evaluation was made 14 days aftertreatment. Injury was scored on a 0-9 scale, 0 representing no injuryand 9 representing plant death. The data presented in FIG. 3Ademonstrate that Madsen1 has tolerance to imazamox.

Because the tolerance in Madsen1 is due to a mutation in the AHAS enzymerendering it resistant to inhibition by imidazolinone herbicides, the invitro activity of AHAS extracted from wild type plants (not having themutation for tolerance) can be compared to the in vitro activity of AHASextracted from tolerant plants in the presence of varying concentrationsof an imidazolinone (IMI) herbicide. Madsen1 was compared to the wildtype variety Madsen. The results are shown in FIG. 3B. FIG. 3B showsthat as the concentration of imazamox increases, the uninhibited AHASenzyme activity decreased faster in wild type lines than in Madsen1. At100 μM imazamox, the residual uninhibited AHAS is sufficient to providea herbicide tolerant response in Madsen1.

Example 4

Feedback Inhibition of AHAS Enzyme Activity by Leucine and Valine

AHAS is known to be feedback inhibited by the branched chain aminoacids. Valine and leucine in combination are especially effectiveinhibitors. When examined, AHAS enzymes extracted from the wild-typevariety Gunner, AP205CL and AP602CL all exhibited comparable patterns ofinhibition by the combination of valine and leucine (FIGS. 4A and 4B).

I claim:
 1. A wheat plant comprising: a) a plant of line Gunner 208, arepresentative sample of seed of the line having been deposited withATCC under Patent Deposit Designation Number PTA-4214, plants of saidline comprising: (i) a Imi2 nucleic acid, said Imi2 nucleic acidcomprising a polynucleotide encoding an IMI2 polypeptide having a serineto asparagine substitution in Domain E as compared to a wild-type AHASpolypeptide, and thereby conferring to said plant, tolerance to 40 gai/ha imazamox; and (ii) a non-mutated wheat AHAS nucleic acid as itsAls1 or Als3 gene and encoding a wild type AHAS polypeptide; or b) aplant obtained by a process comprising: crossing a plant of line Gunner208, a representative sample of seed of the line having been depositedwith ATCC under Patent Deposit Designation Number PTA-4214 with anotherwheat or triticale variety, wherein the plant comprises: (i) a Gunner208 Imi2 nucleic acid, said Gunner 208 Imi2 nucleic acid comprising apolynucleotide sequence encoding an IMI2 polypeptide having a serine toasparagine substitution in Domain E as compared to a wild-type AHASpolypeptide, and thereby conferring to said plant, tolerance to 40 gai/ha imazamox; and (ii) a non-mutated wheat AHAS nucleic acid as itsAls1 or Als3 gene and encoding a wild type AHAS polypeptide.
 2. A plantpart, cell, or seed of the plant of claim 1, wherein said plant part,cell, or seed comprises said Imi2 nucleic acid.
 3. A method forproducing the plant part, cell, or seed of the plant of claim 2,comprising: a) providing a wheat plant; and b) harvesting said plantpart, cell, or seed from said wheat plant, wherein said plant part,cell, or seed comprises said Imi2 nucleic acid.
 4. The seed of claim 2,wherein said seed further comprises a seed treatment.
 5. The seed ofclaim 4, wherein said seed treatment comprises an AHAS-inhibitingherbicide.
 6. The seed of claim 5, wherein said AHAS-inhibitingherbicide comprises an imidazolinone herbicide.
 7. The seed of claim 6,wherein said imidazolinone herbicide comprises at least one of:imazapyr, imazethapyr, imazapic, imazamox, imazaquin, or imazethabenz.8. A method for controlling weeds within the vicinity of a wheat plant,comprising: (a) providing the treated seed of claim 4; and (b) growing awheat plant from said seed; thereby controlling the weeds in thevicinity thereof.
 9. A method for controlling weeds within the vicinityof a wheat plant, comprising: (a) growing, the wheat plant of claim 1;and (b) contacting said plant and weeds in the vicinity thereof with aneffective amount of an AHAS-inhibiting herbicide to which the plant istolerant; thereby controlling the weeds.
 10. The method of claim 9,wherein said AHAS-inhibiting herbicide comprises an imidazolinoneherbicide.
 11. The method of claim 10, wherein said imidazolinoneherbicide comprises at least one of: imazapyr, imazethapyr, imazapic,imazamox, imazaquin, or imazethabenz.
 12. The plant of claim 1, whereinsaid plant comprises multiple IMI nucleic acids wherein at least one ofthe IMI nucleic acids is said Triticum aestivum B-genome Imi2 nucleicacid.
 13. A method for identifying the presence of an Imi2 nucleic acidin a plant, plant part, cell or seed thereof, comprising: (a) providinga plant, plant part, cell, or seed of the plant of claim 1; (b)extracting nucleic acid material from said plant, plant part, cell, orseed; (c) assaying the extracted nucleic acid material for the presenceof the Imi2 nucleic acid, wherein said Imi2 nucleic acid comprises: (i)a nucleic acid comprising the polynucleotide sequence of SEQ ID NO:12;or (ii)a nucleic acid encoding an IMI polypeptide comprising the aminoacid sequence of SEQ ID NO:14.
 14. The method of claim 13, wherein saidassaying step (c) comprises a probe directed to said Imi2 nucleic acid.15. The method of claim 14, wherein said method comprises amplificationof the genetic material by PCR.
 16. The method of claim 10, wherein theimidazolinone herbicide comprises imazethapyr.
 17. The method of claim10, wherein the imidazolinone herbicide comprises imazamox.
 18. A methodfor treating a seed comprising: a. providing the seed of claim 2; and b.applying an herbicidal composition comprising an imidazolinoneherbicide.
 19. The method of claim 18, wherein said imidazolinoneherbicide comprises at least one of: imazapyr, imazethapyr, imazapic,imazamox, imazaquin, or imazethabenz.
 20. The wheat plant of claim 1obtained by the process comprising: crossing a plant of line Gunner 208,a representative sample of seed of the line having been deposited withATCC under Patent Deposit Designation Number PTA-4214 with another wheator triticale variety, wherein the plant comprises: (i) said Gunner 208Imi2 nucleic acid comprising a polynucleotide sequence encoding an IMI2polypeptide having a serine to asparagine substitution in Domain E ascompared to a wild-type AHAS polypeptide, and thereby conferring to saidplant, tolerance to 40 g ai/ha imazamox ; and (ii) said non-mutatedwheat AHAS nucleic acid as its Als1 or Als3 gene and encoding a wildtype AHAS polypeptide.